The strength, or efficacy, of vertebrate synapses is dynamically regulated both during development and in adulthood. A key element of strength is the number of transmitter-containing vesicles released by the presynaptic terminal. These vesicles must be reclaimed, or endocytosed by the terminal and processed for re-release. This project examines endocytosis and vesicle processing with the aim of determining how these steps influence transmitter release and thus synaptic strength. The project utilizes activity-dependent endocytic probes plus a genetically altered protein, synaptopHluorin, to label endocytosed structures, particularly endosomes, for study throughout the processing cycle. The probes are imaged by deconvolution-assisted confocal microscopy and by transmission electron microscopy;both techniques yield three-dimensional renderings of individual terminal boutons at high resolution. The renderings permit endocytosed structures to be computer-analyzed and precisely tracked throughout the cycle. In addition, the project employs electrophysiological recording from postsynaptic sites. The recordings allow direct comparison of synaptic strength to endosome and vesicle behavior as the latter are manipulated under various experimental conditions. Many neurological diseases are characterized by diminished or excessive synaptic strength. Knowledge of how vesicle and endosome processing affect synaptic strength will reveal potential avenues for intervention in these disease states. PUBLIC HEALTH RELEVANCE: Many diseases that affect movement (for example, Parkinson's, Lambert-Eaton's) or mental health (for example, addiction, depression, schizophrenia) are characterized by inappropriate strength, or efficacy, of synapses within the nervous system. This project examines one of the underlying mechanisms that determine strength;its goal is to identify sites for pharmacological intervention, and ultimately to develop new classes of drugs that help correct inappropriate synaptic strength in disease.